Leukotoxin (LtxA/Leukothera) induces ATP expulsion via pannexin-1 channels and subsequent cell death in malignant lymphocytes.

Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium tuberculosis, and Complexity of Antibody Specificities toward This Antigen.

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1-coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM.

Influenza A polymerase subunit PB2 possesses overlapping binding sites for polymerase subunit PB1 and human MAVS proteins.

Influenza A virus is an important human pathogen accounting for widespread morbidity and mortality, with new strains emerging from animal reservoirs possessing the potential to cause pandemics. The influenza A RNA-dependent RNA polymerase complex consists of three subunits (PA, PB1, and PB2) and catalyzes viral RNA replication and transcription activities in the nuclei of infected host cells. The PB2 subunit has been implicated in pathogenicity and host adaptation. This includes the inhibition of type I interferon induction through interaction with the host's mitochondrial antiviral signaling protein (MAVS), an adaptor molecule of RIG-I-like helicases. This study reports the identification of the cognate PB2 and MAVS interaction domains necessary for complex formation. Specifically, MAVS residues 1-150, containing both the CARD domain and the N-terminal portion of the proline rich-region, and PB2 residues 1-37 are essential for PB2-MAVS virus-host protein-protein complex formation. The three α-helices constituting PB2 (1-37) were tested to determine their relative influence in complex formation, and Helix3 was observed to promote the primary interaction with MAVS. The PB2 MAVS-binding domain unexpectedly coincided with its PB1-binding domain, indicating an important dual functionality for this region of PB2. Analysis of these interaction domains suggests both virus and host properties that may contribute to host tropism. Additionally, the results of this study suggest a new strategy to develop influenza A therapeutics by simultaneously blocking PB2-MAVS and PB2-PB1 protein-protein interactions and their resulting activities.

Inhibition of primary effusion lymphoma engraftment in SCID mice by morpholino oligomers against early lytic genes of Kaposi's sarcoma-associated herpesvirus.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The objectives of this study were to investigate the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) against KSHV early lytic genes and to assess their efficacy in severe combined immunodeficiency disease (SCID) mice against PEL engraftment. PPMOs are short, single-stranded DNA analogues that contain a backbone of morpholine rings and phosphorodiamidate linkages and have high delivery efficiency into cells. METHODS: PEL cells were treated with PPMOs against viral interferon regulatory factor 1 (vIRF-1) and expression of vIRF-1 was analysed. PPMOs against vIRF-1 and viral interleukin-6 (vIL-6) were evaluated against PEL cell engraftment in SCID mice. The PPMOs were incubated with BCBL-1 cells and then introduced into the peritoneal cavities of SCID mice, followed by 9 more doses of PPMOs administered at 2-day intervals. At weeks 3 and 9 after BCBL-1 delivery, peritoneal lavage was collected and the ratio of PEL cells among total cells was determined by flow cytometry analysis. RESULTS: Treatment of PEL cells with PPMOs against vIRF-1 led to a reduction of vIRF-1 expression in a dose-dependent manner. Reduction of vIRF-1 expression resulted in higher levels of cellular interferon regulatory factor 3 and of signal transducer and activator of transcription 1. SCID mice receiving a PPMO against vIL-6 had no engraftment of PEL cells and remained healthy throughout the 120-day study. CONCLUSIONS: This study showed that PPMOs can be effective antiviral agents against KSHV. Blocking the expression of early lytic genes might be beneficial for the control of KSHV-associated malignant diseases.

Inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer.

Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the swine industry in many countries, and current control strategies are inadequate. Previously, we explored the strategy of using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to inhibit PRRS virus (PRRSV) replication. PPMOs are nuclease-resistant and single-stranded DNA analogs containing a modified backbone conjugated to a cell-penetrating peptide and can act as antisense agents through steric blockade of complementary messenger RNA. A PPMO (designated 5UP2) targeting highly conserved sequence in the 5'-terminal region of the PRRSV genome was found to produce multi-log10 inhibition of PRRSV replication in cultured cells. To evaluate 5UP2 in vivo, we here administrated the PPMO to 3-week-old piglets via intranasal instillation at 24h before, and 2 and 24h after infection with PRRSV (strain VR2385). Blood samples were collected at 6, 10 and 14 days post-infection (dpi) for detection of PRRSV RNA and antibodies. Necropsy was performed at 14 dpi. Monitoring weight gain in all piglet groups throughout the experiment indicated that PPMO was well tolerated at the doses used. PPMO 5UP2 treatment significantly reduced PRRSV viremia at 6 dpi. On day 14, piglets receiving 5UP2 had significantly less interstitial pneumonia and lower level of anti-PRRSV antibodies than untreated piglets. In alveolar macrophages isolated at the time of necropsy, the expression of antiviral genes in PPMO-treated piglets was elevated in comparison with untreated. This study provides further data indicating that the 5UP2 PPMO can be considered a candidate component for a novel PRRS control strategy.

Recombinant infectious bursal disease virus carrying hepatitis C virus epitopes.

The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector.

Porcine reproductive and respiratory syndrome virus inhibits type I interferon signaling by blocking STAT1/STAT2 nuclear translocation.

Type I interferons (IFNs) IFN-α/ß play an important role in innate immunity against viral infections by inducing antiviral responses. Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the synthesis of type I IFNs. However, whether PRRSV can inhibit IFN signaling is less well understood. In the present study, we found that PRRSV interferes with the IFN signaling pathway. The transcript levels of IFN-stimulated genes ISG15 and ISG56 and protein level of signal transducer and activator of transcription 2 (STAT2) in PRRSV VR2385-infected MARC-145 cells were significantly lower than those in mock-infected cells after IFN-α treatment. IFN-induced phosphorylation of both STAT1 and STAT2 and their heterodimer formation in the PRRSV-infected cells were not affected. However, the majority of the STAT1/STAT2/IRF9 (IFN regulatory factor 9) heterotrimers remained in the cytoplasm of PRRSV-infected cells, which indicates that the nuclear translocation of the heterotrimers was blocked. Overexpression of NSP1ß of PRRSV VR2385 inhibited expression of ISG15 and ISG56 and blocked nuclear translocation of STAT1, which suggests that NSP1ß might be the viral protein responsible for the inhibition of IFN signaling. PRRSV infection in primary porcine pulmonary alveolar macrophages (PAMs) also inhibited IFN-α-stimulated expression of the ISGs and the STAT2 protein. In contrast, a licensed low-virulence vaccine strain, Ingelvac PRRS modified live virus (MLV), activated expression of IFN-inducible genes, including those of chemokines and antiviral proteins, in PAMs without the addition of external IFN and had no detectable effect on IFN signaling. These findings suggest that PRRSV interferes with the activation and signaling pathway of type I IFNs by blocking ISG factor 3 (ISGF3) nuclear translocation.

Morpholino oligomer-mediated protection of porcine pulmonary alveolar macrophages from arterivirus-induced cell death.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes extensive economic losses in the swine industry. Current strategies and vaccines to control the disease are inadequate. We previously demonstrated that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) could potently inhibit PRRS virus (PRRSV) replication in cell cultures. PPMOs are single-stranded DNA analogues containing a modified backbone and cell-penetrating peptide. PPMOs are nuclease-resistant, water-soluble, can enter cells readily and exhibit highly specific binding to complementary RNA. In this study, we examined PPMO-mediated inhibition of PRRSV replication in a primary culture of porcine pulmonary alveolar macrophages (PAMs). METHODS: PAMs were collected from piglets, pre-incubated in culture and infected with PRRSV. Viability, cytopathic effects, virus yield and apoptosis of PAMs in the presence or absence of a PPMO (5UP2) were examined. The 5UP2 PPMO is complementary to a conserved sequence in the 5'-terminal region of the PRRSV genome. The level of several interferon-associated gene products and activity of caspases were monitored. RESULTS: PRRSV infection induced the activity of caspases-3/7, -8 and -9 significantly. Treatment of PAMs with 5UP2 resulted in protection of the cells from PRRSV-induced cell death for at least 7 days and avoided the activation of the caspases evaluated. 5UP2 treatment of PRRSV-infected PAMs also prevented the vigorous induction of interferon-beta and chemokines observed in infected and mock-treated PAMs. CONCLUSIONS: PPMO-mediated suppression of PRRSV replication in PAMs was associated with a reduction of apoptotic and inflammatory responses. These results provide further rationale for the development of PPMO 5UP2 as an antiviral to control PRRSV infection.

Enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers.

Porcine reproductive and respiratory syndrome (PRRS) has caused heavy economic losses in the swine industry worldwide and current strategies to control PRRS are inadequate. Previous studies have shown that peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) can be an effective antiviral against the PRRS virus (PRRSV). PPMO is structurally similar to DNA with modified backbone and is resistant to nuclease. This study was designed to examine increasing inhibitory effect of PPMO combination. Two pairs of PPMOs were identified to have enhanced suppression of PRRSV replication in cell culture, while individual constituents did not work under the same testing conditions. PPMO 5UP1 that is complementary to 5' terminus of PRRSV genome was paired with 4P1 or 7P1 that are complementary to sequence in the translation initiation regions of ORFs 4 and 7, respectively. The PPMO combination also inhibited replication of heterologous strains in the North American PRRSV genotype. Treatment of the cells with the combinations reduced PRRSV RNA and protein levels. In cell-free or cell-based luciferase reporter assays, the PPMO combination suppressed target mRNA translation more effectively than individual constituents, indicating that the suppression was due to their antisense effect. These results suggest potential application of these PPMO combinations for PRRS control.

The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries. The open reading frame 3 (ORF3) of HEV encodes a phosphoprotein with a molecular mass of approximately 13 kDa (hereinafter called vp13). vp13 is essential for establishing HEV infections in animals, yet its exact functions are still obscure. Our current study found evidence showing interaction between vp13 and microtubules. Live-cell confocal fluorescence microscopy revealed both filamentous and punctate distribution patterns of vp13 in cells transfected with recombinant ORF3 reporter plasmids. The filamentous pattern of vp13 was altered by a microtubule-destabilizing drug. The vp13 expression led to elevation of acetylated alpha-tubulin, indicating increased microtubule stability. Its association with microtubules was further supported by its presence in microtubule-containing pellets in microtubule isolation assays. Exposure of these pellets to a high-salt buffer caused release of the vp13 to the supernatant, suggesting an electrostatic interaction. Inclusion of ATP and GTP in the lysis buffer during microtubule isolation also disrupted the interaction, indicating its sensitivity to the nucleotides. Further assays showed that motor proteins are needed for the vp13 association with the microtubules because disruption of dynein function abolished the vp13 filamentous pattern. Analysis of ORF3 deletion constructs found that both of the N-terminal hydrophobic domains of vp13 are needed for the interaction. Thus, our findings suggest that the vp13 interaction with microtubules might be needed for establishment of an HEV infection.

Blockade of viral interleukin-6 expression of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is associated with several malignant disorders, including Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. An early lytic gene of KSHV encodes viral interleukin-6 (vIL-6), a viral homologue of the proinflammatory cytokine and an autocrine/paracrine growth factor human IL-6. In this study, we examined the effects of suppressing vIL-6 expression in PEL cells with antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO). PPMO are ssDNA-analogues that have a modified backbone and enter cells readily. Treatment of PEL cells with a PPMO designed against vIL-6 mRNA led to a marked reduction in the proportion of vIL-6-positive cells detected by immunofluorescence assay. Analysis by Western blot confirmed a specific reduction in the vIL-6 protein level and showed that the reduction was dependent on the dose of vIL-6 PPMO. PEL cells treated with the vIL-6 PPMO exhibited reduced levels of cellular growth, IL-6 expression and KSHV DNA, and an elevated level of p21 protein. Treatment of PEL cells with a combination of two vIL-6 PPMO compounds targeting different sequences in the vIL-6 mRNA led to an inhibitory effect that was greater than that achieved with either PPMO alone. These results show that PPMO targeting vIL-6 mRNA can potently reduce vIL-6 protein translation and indicate that further exploration of these compounds in an animal model for potential clinical application is warranted.

Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome (PRRS) has been devastating the global swine industry for more than a decade, and current strategies to control PRRS are inadequate. In this study we characterized the inhibition of PRRS virus (PRRSV) replication by antisense phosphorodiamidate morpholino oligomers (PMO). Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. PPMO 5UP2 and 5HP are complementary to sequence in the 5' end of the PRRSV genome, and 6P1 and 7P1 to sequence in the translation initiation regions of ORF6 and ORF7, respectively. Treatment of cells with 5UP2 or 5HP caused a 4.5log(10) reduction in PRRSV yield, compared to a control PPMO. Combination of 6P1 and 7P1 led to higher level reduction than 6P1 or 7P1 alone. 5UP2, 5HP, and a combination of 6P1 and 7P1 inhibited PRRSV replication in porcine alveolar macrophages and protected the cells from PRRSV-induced cytopathic effect. Northern blot and real-time RT-PCR results demonstrated that the effective PPMO led to a reduction of PRRSV RNA level. 5UP2 and 5HP inhibited virus replication of 10 other strains of PRRSV. Results from this study suggest potential applications of PPMO for PRRS control.

Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.

Apigenin and cancer chemoprevention: progress, potential and promise (review).

Cancer is one of the major public health burdens in the United States and in other developed countries, causing approximately 7 million deaths every year worldwide. Cancer rates vary dramatically in different regions and populations around the globe, especially between developing and developed nations. Changes in cancer prevalence patterns occur within regions as their populations age or become progressively urbanized. Migration has also contributed to such variations as changes in dietary habits influence cancer rates. These epidemiologic findings strongly suggest that cancer rates are influenced by environmental factors including diet, which is largely preventable. Approaches to prevent cancer include overlapping strategies viz. chemoprevention or dietary cancer prevention. Chemoprevention aims at prevention or reversal of the initiation phase of carcinogenesis or arrest at progression of carcinogenesis through the administration of naturally occurring constituents or pharmacological agents. Cancer prevention through diet may be largely achievable by increased consumption of fruits and vegetables. Considerable attention has been devoted to identifying plant-derived dietary agents which could be developed as promising chemopreventives. One such agent is apigenin. A naturally occurring plant flavone (4', 5, 7,-trihydroxyflavone) abundantly present in common fruits and vegetables including parsley, onions, oranges, tea, chamomile, wheat sprouts and some seasonings. Apigenin has been shown to possess remarkable anti-inflammatory, antioxidant and anti-carcinogenic properties. In the last few years, significant progress has been made in studying the biological effects of apigenin at cellular and molecular levels. This review examines the cancer chemopreventive effects of apigenin in an organ-specificity format, evaluating its limitations and its considerable potential for development as a cancer chemopreventive agent.